Medical Oncology (2019) 36:99 https://doi.org/10.1007/s12032-019-1324-7

ORIGINAL PAPER

Can synthetic lethality approach be used with DNA repair genes for primary and secondary MDS?

Howard Lopes Ribeiro Junior1,2 · Roberta Taiane Germano de Oliveira1,2 · Daniela de Paula Borges1,2 · Marília Braga Costa1,2 · Izabelle Rocha Farias1,2 · Antônio Wesley Araújo dos Santos1,2 · Silvia Maria Meira Magalhães1,2 · Ronald Feitosa Pinheiro1,2,3

Received: 5 August 2019 / Accepted: 15 October 2019 / Published online: 30 October 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract Cancer-specifc defects in DNA repair pathways create the opportunity to employ synthetic lethality approach. Recently, GEMA (gene expression and mutation analysis) approach detected insufcient expression of BRCA or NHEJ (non-homol- ogous end joining) to predict PARP inhibitors response. We evaluated a possible role of DNA repair pathways using gene expression of single-strand break (XPA, XPC, XPG/ERCC5, CSA/ERCC8, and CSB/ERCC6) and double-strand break (ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6, LIG4) in 92 patients with myelodysplastic syndrome (73 de novo, 9 therapy- related (t-MDS). Therapy-related MDS (t-MDS) demonstrated a signifcant downregulation of axis BRCA1-BRCA2-RAD51 comparing to normal controls (p = 0.048, p = 0.001, p = 0.001). XRCC6 showed signifcantly low expression in de novo MDS comparing to controls (p = 0.039) and for patients who presented chromosomal abnormalities (p = 0.047). Downregula- tion of LIG4 was consistently associated with poor prognostic markers in de novo MDS (hemoglobin < 8 g/dL (p = 0.040), neutrophils < 800/mm3 (p < 0.001), patients with excess of blasts (p = 0.001), very high (p = 0.002)/high IPSS-R (p = 0.043) and AML transformation (p < 0.001). We also performed an evaluation of GEPIA Database in 30 cancer types and detected a typical pattern of downregulation as here presented in primary or secondary MDS. All these results suggest synthetic lethality approach can be tested with DNA repair genes (beyond that of BRCA1/2 status) for de novo and therapy-related myelodysplastic syndrome and may encourage clinical trials evaluating the use of PARP1 inhibitors in MDS.

Howard Lopes Ribeiro Junior and Roberta Taiane Germano de Oliveira have equal credits.

Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s1203​2-019-1324-7) contains supplementary material, which is available to authorized users.

* Ronald Feitosa Pinheiro Silvia Maria Meira Magalhães [email protected]; [email protected] [email protected] Howard Lopes Ribeiro Junior 1 Cancer Cytogenomic Laboratory, Center for Research [email protected] and Drug Development (NPDM), Federal University Roberta Taiane Germano de Oliveira of Ceara, 1000 Coronel, Nunes de Melo St. Rodolfo Teóflo, [email protected] Fortaleza, Ceara 60430‑275, Brazil Daniela de Paula Borges 2 Post‑Graduate Program in Medical Science, Federal [email protected] University of Ceara, Fortaleza, Ceara, Brazil Marília Braga Costa 3 Post‑Graduate Program of Pathology, Federal University [email protected] of Ceara, Fortaleza, Ceara, Brazil Izabelle Rocha Farias [email protected]

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Graphic Abstract

Keywords Myelodysplastic syndrome · Synthetic lethality · DNA repair · Gene expression

Introduction Nucleotide excision repair (NER) is the main pathway used by mammals to restore SSB of DNA, removing bulky Primary (or de novo) myelodysplastic syndrome (MDS) is DNA lesions done by environmental mutagens, cancer a hematopoietic stem-cell (HSCs) disorder characterized by chemotherapeutic adducts and UV light [10, 11]. The two bone marrow failure related to aging with increased risk of main branches of NER pathway are the global genome acute myeloid leukemia (AML) transformation [1]. Second- repair (GGR), probing the genome for strand distortions, ary or therapy-related myelodysplastic syndrome (t-MDS) is and the transcription-coupled repair (TCR) that removes a subcategory of therapy-related myeloid neoplasms (t-MNs) distorting lesions that block elongating RNA polymerases. derived from cytotoxic therapies (i.e. chemotherapy and/ Genes of Xeroderma pigmentosum group, especially XPA, or radiotherapy) characterized by complex chromosomal XPC, XPD, XPG [11, 12] and Cockayne syndrome genes, abnormalities and higher risk of progression to AML than especially CSA/ERCC8 and CSB/ERCC6, are essential to de novo MDS [2, 3]. Chemotherapy and radiotherapy induce NER properly function [13, 14]. For both branches of NER DNA lesions in single or double-strands of DNA, predispos- (CGR and TCR), XPA, XPF and XPG/ERCC5 are reported ing to chromosomal rearrangements, amplifcations, dele- as truly efectors. tions, overall genomic instability and cancer development The three main pathways involved in DSBs repair are [3, 4]. homologous recombination (HR), non-homologous end Genomic instability is the hallmark of cancer [5] and joining (NHEJ) [5, 16] and single-strand annealing (SSA) patients with MDS present chromosomal abnormalities and [17]. HR, an error-free system, uses a sister chromatid in mutations in up to 94% of cases [1, 6]. Epidemiologic evi- the formation of heteroduplex [5, 18]. The main proteins dence has suggested up to two-thirds of mutations in cancer are BRCA1 and BRCA2 which interact with recombi- are caused by errors during DNA replication, reinforcing the nase RAD51 to follow the role repair [5, 18]. The NHEJ importance of DNA repair system [7]. For the maintenance mechanism, an error-prone repair, has as main components and protection of genome integrity, cells have molecular Ku80/XRCC5, Ku70/XRCC6 and LIG4. Ku80 and Ku70, DNA repair pathways to restore single (SSB) and double- encoded by XRCC5 and XRCC6 genes, respectively, bind strand (DSBs) breaks of DNA [5]. These breaks have been the DNA ends while LIG4 performs rearrangement, join- associated with chromosomal abnormalities, the most sig- ing the two end-junctions of DNA strands breaks [5, 9, nifcant marker of prognosis for MDS [8, 9]. 19].

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Identifcation of specifc DNA repair pathway defect Table 1 Clinical and laboratory characteristics of de novo MDS can facilitate a precision oncology approach, increasing the patients chance of cure and better therapy selection [20]. Cancer- Variables N % specifc defects in DNA repair pathways create the oppor- tunity to employ synthetic lethality, which has been applied Age (in years) ≤ 60 26 35.6 against cancer cells harboring mutations in BRCA1 and > 60–70 17 23.3 BRCA2 using PARP1 inhibitors (PARPi) [20]. The aim of > 70–80 19 26.0 this report is to evaluate a possible role of specifc defects ≥ 80 11 15.1 related to DNA repair pathways (single (SSB) and double- Gender Male 35 47.9 strand (DSBs) breaks of DNA) in de novo and therapy- Female 38 52.1 related MDS, trying to identify possible targets to synthetic Origin Urban 44 62.9 lethality approach. Rural 26 37.1 Fibrosis Absence 9 47.4 Presence 10 52.6 Materials and methods Nº of dysplasias (BM) 1 12 29.3 2 21 51.2 Patients 3 8 19.5 Dyserythropoiesis Yes 32 78.0 Eighty-two patients with MDS (Seventy-three de novo and No 9 22.0 nine therapy-related MDS patients) were diagnosed at Fed- Dysmegakaryopoiesis Yes 17 41.5 eral University of Ceara (UFC)/Center for Research and No 24 58.5 Drug Development (NPDM) according to WHO 2016. Pri- Dysgranulopoiesis Yes 29 70.7 mary MDS patients were evaluated according to Revised No 12 29.3 International Prognostic Scoring System (IPSS-R). See Micromegakaryocyte Yes 10 21.7 Table 1. Ten bone marrow samples from healthy volunteers No 36 78.3 were used as controls. This study was approved by the Eth- Ring sideroblasts (%) ≥ 1– < 15% 5 22.7 ics Committee of UFC (#1.292.509). Informed consent was > 15– < 50% 6 27.3 ≥ 50% 11 50.0 obtained from all patients and controls. Blasts count (%) ≤ 5% 60 82.2 Cytogenetic analysis > 5%– ≤ 10% 5 6.8 > 10% 8 11.0 Cytogenetic Normal 29 56.9 Conventional G-banding karyotype of mononuclear bone Abnormal 22 43.1 marrow cells of all patients was performed as previously Number of clonal alterations Normal 29 56.9 reported [21]. Briefy, cultures were established in RPMI 1 14 27.5 1640 medium (Gibco, Grand Island, NY, USA) containing 2 4 7.8 30% fetal calf serum. For the 24-h culture, colcemid was 3 or more 4 7.8 added at a fnal concentration of 0.05 μg/mL for the fnal IPSS-R cytogenetic risk group Very good 1 2.0 30 min of culture. After harvesting, the cells were exposed Good 38 74.5 to a hypotonic KCl solution (0.068 mol/L) and fxed with Intermediate 9 17.6 Carnoy bufer fxative (acetic acid/methanol in a 1:3 propor- Poor 0 0.0 tion). The slides were prepared and stained using Giemsa Very poor 3 5.9 solution. Twenty metaphases were analyzed whenever pos- Hemoglobin (g/dL) ≥ 10 17 23.3 sible. The karyotype was prepared using CytoVision Auto- ≥ 8– < 10 20 27.4 mated Karyotyping System (Applied Imaging, San Jose, CA, < 8 36 49.3 USA) and described according to the International System ANC (× 10L−1) ≥ 800 48 65.8 for Human Cytogenetic Nomenclature 2016 [22]. < 800 25 34.2 3 Total RNA extraction Platelet ­(mm ) ≥ 100.000 35 47.9 ≥ 50.000– < 100.000 16 21.9 < 50.000 22 30.1 The bone marrow mononuclear cells were separated after Nº of cytopenias 1 39 53.4 lysis of red cells. Total RNA extractions from isolated mono- 2 18 24.7 nuclear cells (bone marrow), obtained from MDS patients, 3 16 21.9 were performed with TRizol Reagent™ (Invitrogen,

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Table 1 (continued) CA, USA) were used to quantify mRNA expression. Beta- B2 M Variables N % 2-microglobulin gene ( , Hs99999907_m1) and ubiq- uitin (UBC, Hs00824723_m1) were used as endogenous WHO 2016 category MDS-SLD 9 12.3 to normalize diferences in input cDNA. Each sample was MDS-RS 12 16.4 performed in duplicate and the expression ratios were cal- Cq MDS-MLD 39 53.4 culated using ­2−∆ method [23, 24]. MDS-EB-1 4 5.5 MDS-EB-2 9 12.3 IPSS-R risk group Very low 7 13.5 Gene expression profle using GEPIA database Low 31 59.6 Intermediate 6 11.5 Gene expression profle from GEPIA (Gene Expression Pro- High 3 5.8 fling Interactive Analysis) [25] database was selected and Very high 5 9.6 tumor/normal diferential expression levels for each gene WPSS risk group Very low 6 11.8 was conducted via GEPIA tool. GEPIA is a web-based tool Low 19 37.3 (http://gepia.cance​ r-pku.cn/​ ) of RNA sequencing data based Intermediate 16 31.4 on TCGA Research Network (The Cancer Genome Atlas High 7 13.7 Program) (https​://www.cance​r.gov/tcga.) and GTEx (The Very high 3 5.9 Genotype-Tissue Expression) databases (https​://gtexp​ortal​ Transfusion dependence Yes 38 55.9 .org/home). No 30 44.1 Death Yes 23 45.1 No 28 54.9 Statistical analysis AML evolution Yes 9 13.2 No 59 86.8 Data on relative mRNA expression (∆Cq values − quantita- MDS-SLD Myelodysplastic syndrome with single lineage dysplasia, tive cycle) was expressed as mean and range (maximum and MDS-RS myelodysplastic syndrome with ring sideroblasts, MDS- minimum) to determine the possible association between MLD Myelodysplastic syndrome with multilineage dysplasia, MDS- gene expressions and the variables. Normality was evaluated EB Myelodysplastic syndrome with excess blasts, AM Acute myeloid leukemia, WPSS WHO classifcation-based Prognostic Scoring Sys- by Shapiro–Wilk test. Outliers were removed. The Student’s tem, WHO World Health Organization t test or one-way ANOVA with Tukey/Games Howell post hoc test was used when normality was detected. Homoge- neity of variances was tested by Levene’s test. Pearson’s correlation test was used for obtaining the r and the r-square Carlsbad, CA, USA), according to the manufacturer’s ­(r2) values. protocol.

Quantitative real‑time PCR Results Twelve genes of DNA single-strand break repair (SSBR) (XPA, XPC, XPG/ERCC5, CSA/ERCC8, and CSB/ERCC6) Patients and DNA double-strand break repair (DSBR) (ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6, and LIG4) Seventy-three adults with de novo MDS (nine MDS with were evaluated. Quantitative real-time PCR (qPCR) reac- single lineage dysplasia, 12 MDS with ring sideroblasts tions were based on TaqMan® methodology (Applied (MDS-RS), 39 MDS with multilineage dysplasia, and 13 Biosystems, Carlsbad, CA, USA) on 7500 Fast System® MDS with excess blasts) (Table 1) and nine therapy-related (Applied Biosystems, Carlsbad, CA, USA). Pre-developed MDS were evaluated according to WHO 2016. TaqMan gene expression assays for ATM (Hs01112344_m1), The mean age of the patients with de novo MDS and BRCA1 (Hs01556191_m1), BRCA2 (Hs01037423), RAD51 t-MDS was 63.3 (range 22–91) and 61.7 (range 26–87) years (Hs00947967_m1), XRCC5 (Hs00897854_m1), XRCC6 old, respectively. The majority of primary cases were clas- (Hs00750856_s1), LIG4 (Hs00934061_m1), ERCC8/ sifed as good prognosis according to IPSS-R (31/59.6%). CSA (Hs01122124_m1), ERCC6/CSB (Hs00972920_m1), Dyserythropoiesis, dysgranulopoiesis and dysmegakary- ERCC5/XPG (Hs01557031_m1), XPA (Hs00166045_m1) opoiesis were detected in 78.6%, 71.4% and 42.9% of cases, and XPC (Hs01104213_m1) as well as TaqMan Universal respectively. Cytogenetic evaluation of bone marrow cells Master Mix II, with UNG® (Applied Biosystems, Carlsbad, was performed for all cases. See Tables 1 and 2.

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Table 2 Clinical and laboratory characteristics of t-MDS patients Case Treatment Age (years) Karyotype Hb (g/dL) ANC (× 109L) Plateletes (/mm3) RS (%) Blasts (%)

1 Antraciclin 64 46,XY[9] 13.20 775 84.800 0 < 5% 2 AZA 74 No metaphase 7.80 2807 232.000 0 < 5% 3 87 46,XX[20] 12.40 765 112.000 0 ≥ 5% 4 CHOP 71 175,XXXXXXXX,-5,-6,-7,-8,-9,-11,- 11.40 3365 14.900 0 < 5% 13,-14[4]/46,XX,del(5)(q15q33) [8]/46,XX[19] 5 CHOP 77 No metaphase 8.66 976 175.000 0 < 5% 6 Unknown 36 No metaphase 7.00 1800 110.000 0 < 5% 7 Unknown 40 No metaphase 8.00 1900 33.000 0 < 5% 8 AZA 26 46,XX,del(17)(q11.2)93]/46,XX[4] 6.00 3780 300.000 0 < 5% 9 CHOP 81 46,XX[20] 10.90 4949 171.000 53 < 5%

The presence of peripheral cytopenia was considered when Hb < 8 g/dL, ANC count < 800 × 109L and platelets count < 100.000/mm3. High per- centage of blasts was considered when more than 5%. High percentage of ring sideroblasts was considered when more than 15%. Bold text refers to the presence of peripheral cytopenias and/or high percentage of ring sideroblasts and blast

The axis BRCA1‑BRCA2‑RAD51 is downregulated 0.005659–0.017694) expression in therapy-related MDS in therapy‑related MDS comparing to normal controls (Fig. 1a and b; Supple- mentary fle 1). High correlations were detected between There was a signifcant decrease in BRCA1 (Fold= − 1.441; BRCA1 and RAD51 (r = 0.935; p = 0.001), BRCA2 and p = 0.048; 95% IC 0.000022–0.004246), BRCA2 ERCC6 (r = − 0.894; p = 0.003), BRCA2 and XRCC5 (Fold= − 2.184; p = 0.001; 95% IC 0.001332–0.003449), (r = − 0.907; p = 0.002) (Supplementary fle 4). and RAD51 (Fold= − 1.911; p = 0.001; 95% IC

Fig. 1 Expression of DNA repair genes in therapy-related MDS. Downregulation of BRCA1 (Fold= − 1.441), BRCA2 (Fold= − 2.184), Scatter plots demonstrating gene expression and fold-change values and RAD51 (Fold= − 1.911) expression was signifcantly in therapy- of all twelve DNA repair genes in total bone marrow samples of ther- related MDS patients when compared to normal controls apy-related MDS patients. Each graphic represents a gene. a and b

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XRCC6 gene is downregulated in de novo MDS Correlations of the mRNA expressions of DNA repair patients genes in de novo MDS patients

We identified a significant decrease in XRCC6 (p = 0.039; By Pearson’s correlation analysis, we observed signifcant 95% IC 0.000086–0.002660) expression in patients with and positive correlations between genes of single-strand de novo MDS comparing to controls (Fig. 2a and Sup- break repair (SSBR) mechanisms (XPA, XPC, XPG/ERCC5, plementary file 2). CSA/ERCC8, and CSB/ERCC6) and double-strand break repair (DSBR) mechanisms (ATM, BRCA1, BRCA2, RAD51, XRCC5, XRCC6, LIG4) in de novo MDS (Supplementary Chromosomal abnormalities are associated fle 3). with downregulation of DNA repair genes in de High correlations (Pearson’s r > 0.5) were identified novo MDS between XPA and XPC (r = 0.518; p = 0.000), XPA and XPG (r = 0.622; p = 0.000), XPC and ERCC5 (r = 0.518; Patients with de novo MDS who presented chromo- p = 0.000), ERCC5 and XRCC5 (r = 0.692; p = 0.000), somal abnormalities showed low expression of ERCC8 XRCC5 and BRCA1 (r = 0.655; p = 0.000), XRCC5 and (p = 0.047; 95% IC 0.000001–0.000124) (Fig. 2b) RAD51 (r = 0.709; p = 0.000), BRCA1 and BRCA2 (r = 0.547; and XRCC6 (p = 0.05; 95% IC − 0.000004–0.000798) p = 0.000) and BRCA1 and RAD51 (r = 0.809; p = 0.000) (Fig. 2c) when compared to de novo MDS patients with- (Supplementary fle 3). These results reinforce these genes out chromosomal abnormalities (Supplementary file 2). work in a dependent manner as a cascade of events in MDS.

DNA repair gene expression profle in several Downregulation of LIG4 expression is consistently cancers associated with poor prognostic markers in de novo MDS We conducted a detailed cancer versus normal analysis of the DNA repair gene expression in 30 cancer types (GEPIA Severe anemia (hemoglobin < 8 g/dl according to IPSS- database), including acute myeloid leukemia (Supplemen- R) (p = 0.040; 95% CI 0.000017–0.000779) (Fig. 3a) tary fle 5). We observed mRNA expression of BRCA1, and severe neutropenia (neutrophils < 800/mm3 accord- RAD51 and XRCC6 genes were downregulated in leuke- ing to IPSS-R) (p < 0.001; 95% IC 0.000368–0.000868) mia when compared to normal tissues. The same genes are (Fig. 3b) were associated with low expression of LIG4 upregulated in other cancers when compared to normal tis- (Supplementary file 2). sues: adrenocortical carcinoma (ACC), bladder urothelial Patients with excess of blasts (MDS-EB) carcinoma (BLCA), breast invasive carcinoma (BRCA), showed low expression of LIG4 (p = 0.001; 95% IC cervical squamous cell carcinoma and endocervical adeno- 0.000244–0.001003) (Fig. 3c) comparing to initial forms carcinoma (CESC), cholangio carcinoma (CHOL), colon of MDS (MDS-SLD, MDS-RS, MDS-MLD). adenocarcinoma (COAD), lymphoid neoplasm difuse large Patients who transformed into AML pre- B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), sented downregulation of LIG4 (p < 0.001; 95% IC glioblastoma multiforme (GBM), head and neck squamous 0.000653–0.000988) (Fig. 3e) (Supplementary file 2) cell carcinoma (HNSC), kidney chromophobe (KIRC), kid- (Fig. 3e). ney renal papillary cell carcinoma (KIRP), acute myeloid

Fig. 2 Expression of XRCC6 in de novo MDS. Scatter plots demon- in de novo MDS patients when compared to normal controls. b and c strating gene expression of XRCC6 gene of de novo MDS patients. Downregulation of ERCC8 and XRCC6 expression were observed in a Downregulation of XRCC6 expression was signifcantly identifed MDS patients with abnormal Karyotype, respectively

1 3 Medical Oncology (2019) 36:99 Page 7 of 10 99 leukemia (LAML), brain lower grade glioma (LGG), liver and DNA-dependent protein kinase–mediated non-homol- hepatocellular carcinoma (LIHC), lung adenocarcinoma ogous end joining, whereas DNA repair pathways medi- (LUAD), lung squamous cell carcinoma (LUSC), ovarian ated by PARP1 serve as backups. Of utmost importance, serous cystadenocarcinoma (OV), pancreatic adenocar- DNA-PK– mediated non-homologous end-joining-defcient cinoma (PAAD), pheochromocytoma and paraganglioma quiescent leukemia cells and BRCA/DNA-PK–defcient pro- (PCPG), prostate adenocarcinoma (PRAD), rectum adeno- liferating leukemia cells were sensitive to PARP1 inhibitors. carcinoma (READ), sarcoma (SARC), skin cutaneous mela- The central idea of GEMA was to detect at least one gene noma (SKCM), stomach adenocarcinoma (STAD), testicu- could present insufcient expression of BRCA or NHEJ lar germ cell tumors (TGCT), thyroid carcinoma (THCA), to predict response to PARP inhibitors. Regarding NHEJ, thymoma (THYM), uterine corpus endometrial carcinoma downregulation of LIG4 expression was detected in de novo (UCEC), uterine carcinosarcoma (UCS) (Supplementary fle MDS patients here reported and was consistently associ- 5A, 5B, 5C). ated with poor prognostic markers. Using GEMA approach, RAD54−/− and LIG4−/− human pre-B leukemia cell line Nalm6 was sensitive to Olaparib and BMN673, another Discussion PARP1 inhibitor. The response was detected in ki67-qui- escent cells and ki67 + proliferating subpopulation, demon- Synthetic lethality approach is a cell death process that strating the synthetic lethality approach in NHEJ defcient results from alterations in two or more genes while alteration cells with low-LIG4 expression [29]. of either gene alone is not sufcient for cell death. Synthetic Patients with de novo MDS who presented chromosomal lethality between BRCA1 and PARP1 has been explored abnormalities demonstrated low expression of one gene through treatment of homologous recombination defcient related to SSB repair (ERCC8) and other gene related to tumor cells with PARP inhibitors (PARPi). PARP1 is a pro- DSB repair (XRCC6). Low expression of XRCC6 was also tein that plays a major role in base excision repair (BER), identifed in de novo MDS cases when compared to con- required for repairing single-strand breaks. When PARP1 is trol group. The estimated numbers of single-strand breaks inhibited, it results in trapping of PARP1 in DNA, thereby and spontaneous base losses in nuclear DNA are as high preventing downstream repair proteins accessing the dam- as 10,000 per cell per day and, considering other types of age and the initial lesion can be converted to double-strand spontaneous damage, the total may up to 10,0000 per cell breaks of DNA. If BRCA1/BRCA2 do not act properly, the per day [5]. If not properly corrected by SSB system, these double-strand breaks lead to cell death. This approach has lesions may ultimately progress to double-strand breaks been demonstrated as very efective for breast and ovarian of DNA [5]. If double-strand breaks of DNA are not cor- cancers with BRCA1 and BRCA2 mutations [20, 26]. rected, the result can be a chromosomal alteration (i.e. dele- Recently, FDA has granted PARP inhibitor Ruca- tions and translocations) [15]. These results (low ERCC8 parib (Rubraca®) a breakthrough therapy designation for and XRCC6 expressions) link SSB and DSB mechanisms to advanced prostate cancer with BRCA​ mutations. In our cytogenetic abnormalities in primary MDS, reinforcing the study, therapy-related MDS, a very distinctive group of concept DNA repair genes act as a cascade of events. The MDS, presented downregulation of BRCA1-BRCA2-RAD51 low expression of ERCC8 reduces the capability of SSB axis, the most important genes of homologous recombi- repair, predisposing cell to double-strand breaks. If these nation. PARP1 may decrease or prevent accumulation of DSBs are not properly corrected (by the low expression of potentially lethal DSBs either by stimulation of base exci- XRCC6), a chromosomal abnormality may emerge. Accord- sion repair and protein MRE11-mediated recruitment of the ing to GEMA approach, Olaparib exerted strong inhibitory DNA damage marker RAD51 to promote stalled replica- activity against B-NHEJ, and also modestly diminished tion fork restart [27, 28]. t-MDS cases here presented also total NHEJ in XRCC6−/− murine embryonic stem cells showed downregulation of RAD51, increasing the chance (mESCs), but did not afect repair in XRCC6+/+ cells [29]. of PARP inhibitors be more efective than cases with only These results show XRCC6 defcient cells can be sensitive downregulation of BRCA1 or BRCA2 as solid tumors. These to synthetic lethality approach. results suggest synthetic lethality approach may be an option Synthetic lethality approach has been consistently used of treatment for t-MDS, a disorder of grim prognosis without for treating defcient BRCA1/2 solid tumors, fundamental efective options for prolonging survival. genes for properly homologous recombination [30]. The Using a personalized medicine approach called GEMA, low expression of BRCA1-BRCA2-RAD51 in therapy- gene expression and mutation analysis, Nieborowska-Skor- related MDS suggests the same approach for hematopoi- ska et al. (2017) demonstrated leukemia cells accumulate etic stem-cell disorders such as MDS and AML. Reinforc- highly lethal DNA double-strand breaks that are commonly ing the GEMA concept to predictive synthetic lethality repaired by BRCA-dependent homologous recombination to NHEJ in AML cells, results here presented of de novo

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MDS patients showed low expression of XRCC6 and LIG4, be tested with DNA repair genes (beyond that of BRCA1/2 very important components to properly function of NHEJ. status) for primary and therapy-related myelodysplastic All these results suggest synthetically lethal approach can

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◂Fig. 3 Clinical and laboratory features of LIG4 expression in de Funding This study was partially supported by the National Coun- novo MDS. Scatter plots demonstrating gene expression of de novo cil of Technological and Scientifc Development (CNPq) (Grant Nos. MDS patients categorized by laboratory variables (i.e. hemoglobin #420501/2018-5 and #424542/2016-1). and ANC count), clinical variables, classifcation (WHO 2016 clas- sifcation and R-IPSS, respectively) and disease progression (death Compliance with ethical standards or AML evolution), a and b Diferential LIG4 gene expression in MDS patients stratifed by hemoglobin (g/dL) and ANC (× 10L−1) Conflict of interest count, respectively. Low expression of LIG4 gene was observed The authors declare that they have no competing in MDS patients with low count of hemoglobin (8 g/dL) and ANC interest. (< 800 × 10L-). c and d LIG4 gene expression in bone marrow sam- Ethical approval ples of de novo MDS patients stratifed by WHO 2016 classifca- All procedures were approved by the Ethics Commit- tion IPSS-R score prognosis, respectively. Downregulation of LIG4 tee of UFC (#1.292.509) and are in accordance with the 1964 Helsinki gene was identifed in MDS-EB1 and MDS-EB2 cases and in MDS declaration and its later amendments. patients with high and very high risk prognosis. e Downregulation Informed consent of LIG4 gene was identifed in MDS patients who transformed into Informed consent was obtained from all individual AML participants included in the study.

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